Project Summary Proteases represent one of the largest and most well characterized families of enzymes in the human genome. Furthermore, there are many human health conditions such as cancer that are associated with alterations in protease activity and function. Therefore, specific molecular probes that allow individual protease activities to be imaged during disease progression in vivo would both be transformative in our understanding of the roles of proteolytic events that contribute to disease pathology while also providing a direct methods for early disease monitoring and response to therapy. The past decade has produced many diverse classes of molecular probes that can be used for imaging applications. Perhaps one of the most powerful of these reagents is the activity based probe (ABP). However the broad application of ABPs is typically limited by the need to painstakingly optimize probes using synthetic chemistry and often probes lack absolute specificity for a given target enzyme. This proposal will focus on establishing an innovative technology that will allow rapid design of ABPs with exceptional specificity for any given protease target of interest. This will involve application of a phage display method to screen diverse libraries of chemically constrained bi-cyclic peptides linked to a protease reactive electrophile to iteratively screen for covalent binding elements with high potency and selectivity. We propose to establish and validate the phage screening method using two stromal-cell derived protease targets, cathepsin S (cat S) and fibroblast activation protein (FAP), involved in key aspects of tumorigenesis. These new probes will then be validated for imaging applications in mouse models of cancer. The technology developed in this proposal will result in not only a new general method for protease ABP development but also will produce probes with potential future clinical applications in cancer imaging.